Mercedes Fernández-Pascual

Essentiality of boron for symbiotic dinitrogen fixation in pea (Pisum sativum) Rhizobium nodules

The effect of boron deficiency on symbiotic nitrogen fixation in pea (Pisum sativum) was examined. The absence of boron in the culture medium resulted in a decrease of the number of nodules and an alteration of nodule development leading to an inhibition of nitrogenase activity. Examination of boron-deficient nodules showed dramatic changes in cell walls and in both peribacteroid and infection thread membranes, suggesting a role for boron in the stability of these structures. These results indicate that boron is a requirement for normal nodule development and functionality.

A fadD mutant of Sinorhizobium meliloti shows multicellular swarming migration and is impaired in nodulation efficiency on alfalfa roots

Swarming is a form of bacterial translocation that involves cell differentiation and is characterized by a rapid and co‐ordinated population migration across solid surfaces. We have isolated a Tn5 mutant of Sinorhizobium meliloti GR4 showing conditional swarming. Swarm cells from the mutant strain QS77 induced on semi‐solid minimal medium in response to different signals are hyperflagellated and about twice as long as wild‐type cells. Genetic and physiological characterization of the mutant strain indicates that QS77 is altered in a gene encoding a homologue of the FadD protein (long‐chain fatty acyl‐CoA ligase) of several microorganisms. Interestingly and similar to a less virulent Xanthomonas campestris fadD(rpfB) mutant, QS77 is impaired in establishing an association with its host plant. In trans expression of multicopy fadD restored growth on oleate, control of motility and the symbiotic phenotype of QS77, as well as acyl‐CoA synthetase activity of an Escherichia coli fadD mutant. The S. meliloti QS77 strain shows a reduction in nod gene expression as well as a differential regulation of motility genes in response to environmental conditions. These data suggest that, in S. meliloti, fatty acid derivatives may act as intracellular signals controlling motility and symbiotic performance through gene expression.

Differential effectiveness of novel and old legume–rhizobia mutualisms: implications for invasion by exotic legumes

The degree of specialization in the legume–rhizobium mutualism and the variation in the response to different potential symbionts are crucial factors for understanding the process of invasion by exotic legumes and the consequences for the native resident plants and bacteria. The enhanced novel mutualism hypothesis predicts that exotic invasive legumes would take advantage of native rhizobia present in the invaded soils. However, recent studies have shown that exotic legumes might become invasive by using exotic introduced microsymbionts, and that they could be a source of exotic bacteria for native legumes. To unravel the role of novel and old symbioses in the progress of invasion, nodulation and symbiotic effectiveness were analyzed for exotic invasive plants and native co-occurring legumes in a Mediterranean coastal dune ecosystem. Although most of the studied species nodulated with bacteria from distant origins these novel mutualisms were less effective in terms of nodulation, nitrogenase activity and plant growth than the interactions of plants and bacteria from the same origin. The relative effect of exotic bradyrhizobia was strongly positive for exotic invasive legumes and detrimental for native shrubs. We conclude that (1) the studied invasive legumes do not rely on novel mutualisms but rather need the co-introduction of compatible symbionts, and (2) since exotic rhizobia colonize native legumes in invaded areas, the lack of effectiveness of these novel symbiosis demonstrated here suggests that invasion can disrupt native belowground mutualisms and reduce native legumes fitness.

Accumulating behaviour of Lupinus albus L. growing in a normal and a decalcified calcic luvisol polluted with Zn

Lupinus albus L. is a leguminous plant that is starting to generate interest for the phytoremediation soils showing intermediate metal pollution. Among these metals, Zn causes major phytotoxicity problems and is common in polluted soils of central Spain. The purpose of this study was to explore the nutritional behaviour of this plant species towards increasing Zn concentrations in two calcic luvisol soils: a normal basic soil and a decalcified acid soil. For this purpose the effects of different Zn concentrations on mineral nutrition, growth, nodulation and nitrogenase activity of nodulated Lupinus albus cv. Multolupa plants has been investigated. A 12-week trial was performed in pots under greenhouse conditions. In each soil, four replicate pots were set up per treatment (100, 150, 300, 500 and 700 ppm Zn). Seeds were inoculated with a Bradyrhizobium sp. (Lupinus) strain ISLU-16. Statistical analysis of data revealed significant effects of soil Zn on grown, plant mineral composition and nodulation. Lupin growth was better in acid soil than in basic soil with the low dose of Zn applied, although plant growth in acid soil was severely affected from 300 ppm Zn, where the pH of the soil was 4.7. Zn application produce nutritional imbalances, especially with the higher dose added. Most of Zn accumulation occurred in the roots in both types of soils. In acid soil, lupin absorbs high amounts of Zn in both root (4650 ppm) and aerial part (3605 ppm), when the doses of Zn applied was 300 ppm. This feature permits Lupinus albus cv. Multolupa to be considered as potential phytoremediator and also for the revegetation of degraded landfill areas with slightly acid or neutral soils polluted with Zn.

Stress tolerance, genetic analysis and symbiotic properties of root-nodulating bacteria isolated from Mediterranean leguminous shrubs in Central Spain.

Nine root-nodulating bacterial isolates were obtained from the leguminous shrubs Spartium junceum, Adenocarpus hispanicus, Cytisus purgans, Cytisuslaburnuum, Retama sphaerocarpa and Colutea arborescens in areas of Central Spain. A poliphasic approach analyzing phenotypic, symbiotic and genetic properties was used to study their diversity and characterize them in relation to Mediterranean conditions. Stress tolerance assays revealed marked variations in salinity, extreme pH and cadmium tolerance compared with reference strains, with the majority showing salinity, alkalinity and Cd tolerance and three of them growing at acid pH. Variation within the 16S rRNA gene was examined by amplified 16S rDNA restriction analysis (ARDRA) and direct sequencing to show genetic diversity. Phylogeny confirmed the close relationship of four isolates with Bradyrhizobium canariense, three with Phylobacterium myrsinacearum, one with Rhizobium rhizogenes and another with Mesorhizobium huakuii. The cross inoculation tests revealed wide spectra of nodulation. This is the first report of P. myrsinacearum being able to nodulate these leguminous shrubs, and also the first time reported the association between B.canariense, R. rhizogenes and M. huakuii and C. laburnuum, C. purgans and C. arborescens, respectively. These results suggested that native rhizobia could be suitable candidates as biofertilizers and/or inoculants of leguminous shrubs with restoration or revegetation purposes in Mediterranean areas.

Enhancing the early performance of the leguminous shrub Retama sphaerocarpa (L.) Boiss.: fertilisation versus Rhizobium inoculation

We have investigated the effect on growth of fertilisation versus biological nitrogen fixation by rhizobial nodules in Retama sphaerocarpa(L.) Boiss, a leafless leguminous shrub native to the Iberian Peninsula and North-West Africa that has generated interest for revegation of dry Mediterranean habitats. Our main objective was to optimise the formation of root nodules under nursery conditions and to evaluate their influence on the first year of seedling growth in comparison with standard fertilisation. Seedlings of R. sphaerocarpa from two Spanish localities were grown under two levels of fertilisation, and half of each were inoculated with rhizobia isolated from adult Retama, Cytisus and Adenocarpusplants in the field. Although some promiscuity was observed, nodulation was significantly successful with specific rhizobia. At the end of the experiment, highly fertilised plants were taller and heavier and exhibited larger photosynthetic rates than either nodulated or non-nodulated plants under low fertilisation. High fertilisation enhanced seedling growth but inhibited both the nodulation and the nitrogenase activity of the nodules. Thus, physiological differences between nodulated and non-nodulated plants were observed in the low but not in the high fertilisation treatment. Nitrogen uptake and use was enhanced by root nodules, which translated into enhanced photosynthesis and growth. Since inoculation is simple, environmentally friendly and cheap, and nodulated plants are more likely to overcome transplant stress than non-nodulated ones, our results suggest that inoculation together with low, background fertilisation (instead of high fertilisation) should be used when producing high quality seedlings of this autochthonous Mediterranean shrub.

Mercury-resistant rhizobial bacteria isolated from nodules of leguminous plants growing in high Hg-contaminated soils

A survey of symbiotic bacteria from legumes grown in high mercury-contaminated soils (Almadén, Spain) was performed to produce a collection of rhizobia which could be well adapted to the environmental conditions of this region and be used for restoration practices. Nineteen Hg-tolerant rhizobia were isolated from nodules of 11 legume species (of the genera Medicago, Trifolium, Vicia, Lupinus, Phaseolus, and Retama) and characterized. Based on their growth on Hg-supplemented media, the isolates were classified into three susceptibility groups. The minimum inhibitory concentrations (MICs) and the effective concentrations that produce 50% mortality identified the patterns of mercury tolerance and showed that 15 isolates were tolerant. The dynamics of cell growth during incubation with mercury showed that five isolates were unaffected by exposure to Hg concentrations under the MICs. Genetic analyses of the 16S rRNA gene assigned ten strains to Rhizobium leguminosarum, six to Ensifer medicae, two to Bradyrhizobium canariense, and one to Rhizobium radiobacter. Inoculation of host plants and analysis of the nodC genes revealed that most of them were symbiotically effective. Finally, three isolates were selected for bioremediation processes with restoration purposes on the basis of their levels of Hg tolerance, their response to high concentrations of this heavy metal, and their genetic affiliation and nodulation capacity.

Effects of the herbicides Lindex and Simazine on chloroplast and nodule development, nodule activity, and grain yield inLupinus albus L.

Field and laboratory investigations were carried out to determine the influence of two commercial herbicides Lindex and Simazine on symbiotic N2 fixation, the photosynthetic apparatus, percentage of proteins, and grain yield of lupins (Lupinus albus L. cv. Multolupa). The herbicides were added (3 kg commercial product in 9001 per ha) two weeks after sowing. The nodulated roots were tested for nitrogenase activity by the acetylene reduction assay (ARA) at the first and second flowering. The ARA values decreased with herbicide application. The decrease was smaller in the Simazine treatments with inoculated seeds. The ARA for plants treated with Lindex did not increase with inoculation. Nitrogenase activity was greater in the non-inoculated plants growing together with weeds. The effect of Simazine on the photosynthetic apparatus proved to be more toxic than that of Lindex, not only considerably reducing the size of the chloroplasts but also affecting the grana structure. The latter appeared as an electron-dense compact mass. There were no visible alterations in the photosynthetic apparatus of plants treated with Lindex. However starch, which was not present in the control leaves, accumulated.No ultrastructural changes were observed in the nodule cells treated with Lindex. Simazine altered nodule cells by causing vesicle formation, degeneration of bacteria, and by decreasing the number of N2-fixing bacteroids.The grain yield decreased with both herbicide treatments, the decrease being lower in the inoculated plants, especially in the Simazine treatment.Our results suggest that each herbicide had a different effect on N2 fixation, the photosynthetic apparatus and grain yield. Laboratory studies indicated that cyanazine and simazine did not adversely affect the growth of Bradyrhizobium.

Effects of Cyanazine and Linuron on Chloroplast Development, Nodule Activity and Protein Metabolism in Lupinus albus L.

Summary The effects of cyanazine and linuron on development, mesophyll and nodule ultrastructure, nitrogen fixation and protein metabolism of Lupinus albus L. cv. Multolupa inoculated with Bradyrbizobium sp. ( Lupinus ) OR-14, grown under controlled conditions, were investigated. Cyanazine and linuron decreased the weight of both roots and shoots. Cyanazine was more toxic than linuron with regard to the photosynthetic apparatus, nodule ultrastructure, and nitrogen fixation. Concentrations above 4μM cyanazine and 12μM linuron completely inhibited nodulation. The adverse effects on nitrogen fixation due to herbicide application were indirectly caused through action on the photosynthetic apparatus, since these herbicides did not affect the microsymbiont in vitro culture. Leaf protein metabolism was also altered, as determined by SDS-PAGE. When the concentration applied was 20μM cyanazine or 80μM linuron a polypeptide of 17 kDa, which was not found in control plants, appeared. Furthermore, a band of 30 kDa could be observed in the cyanazine treatments. On the other hand, staining of the large subunit of Ribulose-1,5-bisphosphate carboxylase became fainter when the cyanazine and linuron concentrations were increased.

Protective Enzymes against Active Oxygen Species during Nitrate-Induced Senescence of Lupinus albus Nodules

Summary Nitrate application to nodulated legumes inhibits nitrogenase activity and causes the premature senescence of nodules. In this work the activity of antioxidant enzymes and the production of toxic oxygen species during nitrate-induced senescence of Lupinus albus L. cv. Multolupa nodules was investigated. Four-six days of nitrate treatment induced a decrease in the activity of the main enzymes that scavenge H 2 O 2 in the nodule cytosol, catalase and ascorbate peroxidase. Total peroxidase activity increases in response to nitrate treatment, but H 2 O 2 eventually builds up in nodule cytosol. It is suggested that this H 2 O 2 causes the first damage to leghemoglobin apoprotein, which results in a decreased in situ immunolabelling of this hemoprotein. Degradation of Lb continues with the rupture of the heme group after 8 days of nitrate treatment. This may result in the release of catalytic iron, which in turn may promote the formation of hydroxyl radicals from the precursors H 2 O 2 and O 2 , or from H 2 O 2 through a Fenton-type reaction.